Linearized Bradford Assay with B-Lactogloblin as Standard for Use with Snake Venoms
Analyzing the protein content of snake venom, a mixture of proteins, in solution is problematic due to the protein composition of venom being specific to each species and individual. A common method of determining the concentration of a protein in solution is to use the absorbance at 280 nm. The absorbance is due mostly to the presence of the amino acid tryptophan but is also influenced by the presence of tyrosine and cysteine. The problem with using this method is the interference from other chromophores such as DNA or other impurities that absorb near 280 nm. When analyzing snake venom samples purity can be an issue due to venom milking methods. Also the variability of the number and nature of proteins in the venom causes variability in the absorbance at 280 nm. In the linearized Bradford Assay, protein concentration is determined from the absorbance changes that occur when the protein binds a dye (Coomassie brilliant blue). For the linearized assay, the absorbance ratio, 590 nm/450 nm is used to calculate the amount of protein in the assay. The linearized Bradford Assay uses a standard protein and the concentration of the unknown protein is determined by comparison. Bovine Serum Albumin, BSA, is used commonly as a standard due to its ready availability, low cost, and known extinction coefficient. We show here that BSA poorly predicts the amount of snake venom in solution. Our results show that β-Lactogloblin is a better standard for snake venoms while also having a known extinction coefficient.